Compositions and processes for genotyping single nucleotide polymorphisms

Abstract

The invention relates to processes, compositions and kits for analysis of nucleic acid variations, especially single nucleotide polymorphisms (SNPs). An inventive process includes a procedure to enzymatically remove inorganic pyrophosphate from a sample prior to and/or during a single base extension reaction. Processes and compositions described herein are especially useful in nucleic acid analysis methods designed to minimize transfer and separation steps.

1 . A process for inhibiting misincorporation of a terminator in a single base primer extension reaction, comprising: 
 providing a product of a nucleic acid synthesis reaction, the product comprising a nucleic acid template and a quantity of inorganic pyrophosphate;    incubating the product and an inorganic pyrophosphatase under conditions sufficient to decrease the quantity of pyrophosphate, to yield a purified reaction product;    combining the purified reaction product, a primer, a terminator having a detectable label, and a polymerase to form a mixture; and    incubating the mixture under conditions sufficient to extend the primer by addition of the terminator in a single base primer extension reaction, wherein decreasing the quantity of inorganic pyrophosphate in the product of a nucleic acid synthesis reaction inhibits pyrophosphorolysis in the single base primer extension reaction, so as to inhibit misincorporation of a terminator.    
     
     
         2 . The process of  claim 1  wherein the nucleic acid synthesis product further comprises a residual reaction component selected from the group consisting of: a residual primer and a nucleotide.  
     
     
         3 . The process of  claim 2  further comprising: 
 adding an enzyme selected from the group consisting of: an exonuclease, an alkaline phosphatase, and a combination thereof to the nucleic acid synthesis product; and    incubating the nucleic acid synthesis product and enzyme under conditions sufficient to degrade the residual reaction component.    
     
     
         4 . The process of  claim 2  further comprising: 
 adding an enzyme selected from the group consisting of: an exonuclease, an alkaline phosphatase, and a combination thereof to the purified reaction product; and    incubating the nucleic acid synthesis product and enzyme under conditions sufficient to degrade the residual reaction component.    
     
     
         5 . The process of  claim 3  further comprising: 
 inactivating the enzyme.    
     
     
         6 . The process of  claim 1  further comprising inactivating the inorganic pyrophosphatase.  
     
     
         7 . The process of  claim 1  wherein the detectable label is a fluorescent label.  
     
     
         8 . (canceled)  
     
     
         9 . The process of  claim 1  further comprising detecting the detectable label.  
     
     
         10 . The process of  9  wherein the step of detecting the label comprises detection of fluorescence polarization.  
     
     
         11 - 12 . (canceled)  
     
     
         13 . The process of  claim 3  wherein the alkaline phosphatase is selected from the group consisting of: bacterial alkaline phosphatase, calf intestinal alkaline phosphatase and a combination thereof.  
     
     
         14 . The process of  claim 3  wherein the alkaline phosphatase is shrimp alkaline phosphatase.  
     
     
         15 . The process of  claim 3  wherein the exonuclease is selected from the group consisting of: lambda exonuclease, mung bean exonuclease, Bal31 exonuclease, T7 exonuclease and a combination thereof.  
     
     
         16 . The process of  claim 3  wherein the exonuclease is exonuclease I.  
     
     
         17 . (canceled)  
     
     
         18 . The process of  claim 1  wherein the polymerase is a thermostable polymerase having a greater affinity for an acyclo nucleoside terminator than for a dideoxyterminator.  
     
     
         19 - 20 . (canceled)  
     
     
         21 . The process of  claim 1  wherein the steps are performed in a single reaction container.  
     
     
         22 . (canceled)  
     
     
         23 . The process of  claim 1  wherein the terminator is an acyclo nucleoside terminator.  
     
     
         24 . The process of  claim 23  wherein the acyclo nucleoside terminator comprises a detectable label.  
     
     
         25 . (canceled)  
     
     
         26 . A process for inhibiting misincorporation of a terminator in a single base primer extension reaction, comprising: 
 providing a product of a nucleic acid synthesis reaction, the product comprising a nucleic acid template and a quantity of inorganic pyrophosphate;    incubating the product and a pyrophosphate removing enzyme under conditions sufficient to decrease the quantity of pyrophosphate, to yield a purified reaction product;    combining the purified reaction product, a primer, a terminator having a detectable label, and a polymerase to form a mixture; and    incubating the mixture under conditions sufficient to extend the primer by addition of the terminator in a single base primer extension reaction, wherein decreasing the quantity of inorganic pyrophosphate in the product of a nucleic acid synthesis reaction inhibits pyrophosphorolysis in the single base primer extension reaction, so as to inhibit misincorporation of a terminator.    
     
     
         27 . The process of  claim 26  wherein the nucleic acid synthesis product further comprises a residual reaction component selected from the group consisting of: a residual primer and a nucleotide.  
     
     
         28 . The process of  claim 27  further comprising: 
 adding an enzyme selected from the group consisting of: an exonuclease, an alkaline phosphatase, and a combination thereof to the nucleic acid synthesis product; and    incubating the nucleic acid synthesis product and enzyme under conditions sufficient to degrade the residual reaction component.    
     
     
         29 . The process of  claim 27  further comprising: 
 adding an enzyme selected from the group consisting of: an exonuclease, an alkaline phosphatase, and a combination thereof to the purified reaction product; and    incubating the nucleic acid synthesis product and enzyme under conditions sufficient to degrade the residual reaction component.    
     
     
         30 . The process of  claim 26  further comprising inactivating the inorganic pyrophosphatase.  
     
     
         31 . (canceled)  
     
     
         32 . A process for inhibiting misincorporation of a terminator in a single base primer extension reaction, comprising: 
 combining a nucleic acid template, a primer, an inorganic pyrophosphatase, an acyclo nucleoside terminator, and a polymerase to yield a mixture substantially free of deoxynucleotide-triphosphates; and    incubating the mixture under conditions sufficient to extend the primer by addition of the acyclo nucleoside terminator, wherein the pyrophosphatase inhibits pyrophosphorolysis in the single base primer extension reaction, thereby reducing misincorporation of a terminator.    
     
     
         33 - 63 . (canceled)